The present invention relates to a highly spatially resolved method for permeabilisation of cell structures, organelle structures and cell-like structures in order to transfer compounds into or out of the structures. It also relates to use of this method.
During the last two decades there has been a tremendous growth in experimental methods that allow for biochemical and biophysical investigations of single cells. Such methods include patch clamp recordings which can be used for measurement of transmembrane currents through a single ion channel (O. P. Hamill, A. Marty, E. Neher, B. Sakman, F. J. Sigworth, Pfleugers Arch. 391, 85-100 (1981)); laser confocal microscopy imaging techniques that can be used to localise bioactive components in single cells and single organelles (S. Maiti, J. B. Shear, R. M. Williams, W. R Zipfel, W. W. Webb, Science, 275, 530-532 (1997)); use of near field optical probes for pH measurements in the cell interior; and use of ultramicroelectrodes for measurement of release of single catechol- and indol-amine-containing vesicles (R. H. Chow, L. von Ruden, E. Neher, Nature, 356, 60-63 (1992) and R. M. Wightman, J. A. Jankowski, R. T. Kennedy, K. T. Kawagoe, T. J. Scroeder, D. J. Leszczyszyn, J. A. Near, E. J. Diliberto Jr., O. H. Viveros, Proc. Natl. Acad. Sci. U.S.A., 88, 10754-10758 (1991)). Although numerous high-resolution techniques exist to detect, image and analyse the contents of single cells and subcellular organelles, few methods exist to control and manipulate the biochemical nature of these compartments. Most compounds for biological and medical use that are of interest to include in cells are polar. Polar solutes are cell-impermeant and unable to pass biological membranes unless specific transporters exist. Often in experimental biology as well as in biochemical and clinical work, polar solutes need to be administered to the cytoplasm of cells or to the interior of organelles. Examples of such polar solutes are nanoparticles, dyes, drugs, DNAs, RNAs, proteins, peptides, and amino acids. At present, it is extremely difficult, for example, to label a cell in a cell culture with a dye, or transfect it with a gene without labelling or transfecting its adjacent neighbour. It is even more difficult to introduce polar molecules into organelles because of their size which many times is smaller than the resolution limit of a light microscope, or at least less than a few micrometers in diameter.
Microinjection techniques for single cells and single nuclei have also been described (see e.g. M. R. Capecchi, Cell, 22, 479-488 (1980), but these get increasingly difficult to implement as the size of the cell or organelle decreases. For cells and organelles measuring only a few micrometers in diameter or less, microinjection techniques become virtually impossible to use.
It has for a long time been recognised that cell membranes can be permeabilised by pulsed electric fields (see e.g. Zimmermann, U. Biochim. Biophys Acta, 694, 227-277 (1982); Tsong, T. Y. Biophys. J., 60, 297-306 (1991); Weaver, J. C. J. Cell. Biochem., 51, 426-435 (1993)). This technique is called electroporation. The membrane voltage, Vm, at different loci on phospholipid bilayer spheres during exposure in a homogenous electric field of duration t, can be calculated from:
Vm=1.5rcEcosxcex1[1-exp (xe2x88x92xcfx84/ t)]xe2x80x83xe2x80x83(1)
where E is the electric field strength, rc is the cell radius, xcex1, the angle in relation to the direction of the electric field, and xcfx84 the capacitive-resistive time constant. Pore-formation will result at spherical coordinates exposed to a maximal potential shift, which is at the poles facing the electrodes (cosxcex1=1 for xcex1=0; cosxcex1=xe2x88x921 for xcex1=xcfx80). Generally, electric field strengths on the order of from 1 to 1.5 kV/cm for durations of a few xcexcs to a few ms are sufficient to cause transient permeabilisation in 10-xcexcm-outer diameter spherical cells. A recent study shows that isolated mitochondria, because of their correspondingly smaller size, require 7-10-fold higher electric field strengths to incorporate a 7.2-kilobase plasmid DNA (J-M. Collombet, V. C. Wheeler, F. Vogel, and C. Coutelle J. Biol. Chem., 272, 5342-5347 (1997)). Mitochondrial outer-membrane fusion at lower electric field strengths of about 2.5 kV/cm has also been observed.
Traditionally, electroporation is made in a batch mode allowing for administration of polar solutes into several millions of cells simultaneously. The electrodes producing such fields can be several square centimetres and the distance between the electrodes several centimetres, thus requiring high-voltage power sources to obtain the needed electrical field strength to cause electrically induced permeabilisation of biological membranes.
One advantage of electroporation compared to microinjection techniques is that electroporation can be extremely fast, and precisely timed (see e.g. K. Kinosita, K., Jr., I. Ashikawa, N. Saita, H. Yoshimura, H. Itoh, K. Nagayama, and A. Ikegami J. Biophys., 53, 1015-1019 (1988); M. Hibino, M. Shigemori, H. Itoh, K. Nagayama, and K. Kinosita, K., Jr., Biophys. J., 59, 209-220 (1991)) which is of importance in studying fast reaction phenomena.
Instrumentation that can be used for electroporation of a small number of cells in suspension (K. Kinosita, Jr., and T. Y. Tsong, T. Biochim. Biophys. Acta, 554, 479-497 (1979); D. C Chang, J. Biophys., 56, 641-652 (1989; P. E. Marszalek, B. Farrel, P. Verdugo, and J. M. Fernandez, Biophys. J., 73, 1160-1168 (1997)) and for a small number of adherent cells grown on a substratum (Q. A. Zheng, and D. C. Chang, Biochim. Biophys. Acta, 1088, 104-110 (1991); M. N. Teruel, and T. Meyer Biophys. J., 73, 1785-1796 (1997)) have also been described. The design of the electroporation device constructed by Marszalek et al. is based on 6 mm long 80 xcexcm diameter platinum wires that are glued in a parallel arrangement at a fixed distance of 100 xcexcm to a single glass micropipette. The design by Kinosita and Tsong uses fixed brass electrodes spaced with a gap distance of 2 mm, the microporator design of Teruel and Meyer relies on two platinum electrodes that are spaced with a gap distance of about 5 mm, and the electroporation chamber design by Chang uses approximately 1 mm-long platinum wires spaced at a distance of 0.4 mm. It is obvious, that these electroporation devices, which are optimized for usage in vitro, create electric fields that are several orders of magnitude larger than the size of a single cell which typically is 10 xcexcm in diameter, and thus can not be used for exclusive electroporation of a single cell or a single organelle or for electroporation inside a single cell. The techniques do not offer a sufficient positional and individual control of the electrodes to select a single cell, or a small population of cells. Furthermore, these techniques are not optimized for electroporation in vivo or for electroporation of remote cells and tissue. Electroporation devices for clinical and in vivo applications have also been designed. Examples include devices for electroporation-mediated delivery of drugs and genes to tumours (WO 96/39226) and to blood cells (U.S. Pat. No. 5,501,662) and to remote cells and tissue (U.S. Pat. No. 5,389,069). Likewise, these can not be used to create a highly localised electric field for electroporation of a single cell, a single organelle, or a population of organelles within a cell.
One of the major disadvantages with the known techniques is that they are not applicable for permeabilisation of single cells or single intracellular organelles.
The present invention provides a highly spatially resolved technique to alter the biochemical content of single cells and organelles, based on permeabilisation of phospholipid bilayer membranes by pulsed electric fields, i.e. so called electroporation.
An advantage the method according to the present invention compared to known methods for electroporation is that the method according to the invention is characterised by an extremely high spatial resolution defined by highly focused permeabilising electric fields. Such highly focused electric fields are obtained by using a pair of electroporation electrodes with outer diameters in the range of a few manometres to a few micrometers. This enables electroporation of single cells and even intracellular structures. The electrodes are controlled individually with high-graduation micropositioners, thereby enabling precise electrode alignment with respect to a structure to be permeabilised. During the effective pore-open time, cell-impermeant solutes added to the extracellular or extraorganellar medium can enter the cell or organelle interior by diffusion.
In contrast to the known microinjection techniques for single-cells and single nuclei, the present invention can be applied for biological containers of subfemtoliter ( less than 10xe2x88x9215 l) volumes or less than a few micrometers in diameter, which is another important advantage of the invention.
Furthermore, the present invention distinguishes itself from prior art in that electroporation with single-cell or subcellular spatial resolution is accomplished by applying the electric field through nanometer- and micrometer-diameter electrodes with extremely short inter-electrode distances. The electrodes are controlled individually by high-graduation micromanipulators, allowing precise focusing of the electric field between the electrodes. Electroporation of individual cells and individual organelles can thereby be accomplished. Electroporation can with the present invention be performed in such a way that only a target cell is permeabilised and not its adjacent neighbour. Also, individual cellular processes can be electroporated. Even spatially well-defined intracellular domains with a targeted class of organelles can be held under a localised electric field with this invention, thereby enabling transfer of polar solutes into organelles. Applications of electroporation of organelles include alterations of the mitochondrial genome. It is well known that mutations in the mitochondrial genome can lead to a multitude of diseases, and that gene therapy can potentially be of major importance. So far, however, mitochondria has to be isolated from the cells before transfer of the new genefragment into the mitochondria can be performed. Then, the mitochondria has to be reinserted into the cell. The technique according to the invention makes it possible to directly insert genes into the mitochondria when they are contained inside a cell. This is a significant advancement over traditional schemes for transfection of mitochondria.
In addition to the high spatial resolution achieved by using nano- and micro-electrodes, the technique according to the invention avoids the use of expensive high-voltage pulse generators, and complicated microchamber mounts. The method according to the invention can in principle be battery-operated because the spacing between the electrodes is small, typically 20 xcexcm or less, which result in a high electric field strength with a small amplitude voltage pulse. This technique is the first demonstration of selective solute-transfer into biological structures using highly focused electric fields of single-cell and subcellular dimensions.
The method according to the invention can be additionally used for biosensor applications where a cell or a cell-like structure is placed in a permebilising dc or ac electric field while supplemented with drugs or other compounds of interest. A special application is the combination of electroporation and miniaturised chemical separations, where hollow liquid electrodes made of fused silica or similar materials are used.
With ultramicroelectrodes, such as carbon fibre electrodes, controlled by high-graduation micromanipulators, used according to the present invention, it is easy to focus the electrical field to very well-defined regions.
Thus, the present invention relates to a method for permeabilisation of a cell structure consisting of a small population of cells, a single cell, an intracellular structure or an organelle, characterised in that it enables highly spatially resolved permeabilisation and that it comprises the following steps:
(a) at least one microelectrode is provided;
(b) said at least one microelectrode is connected to a power supply;
(c) said at least one microelectrode, individually controlled by high-graduation micromanipulators, is placed close to the cell structure;
(d) a highly focused electric field of a strength sufficient to obtain electroporation is applied between said at least one microelectrode and at least one other electrode also connected to said power supply.
One embodiment of the invention relates to a method for permeabilisation of a cell structure consisting of a single cell or a cell-like structure, an intracellular structure or an organelle characterised in that it comprises the following steps:
(a) microelectrodes are provided;
(b) the microelectrodes are connected to a power supply;
(c) the electrodes, individually controlled by high-graduation micromanipulators, are placed close to the cell or organelle structure or inside a single cell at an appropriate inter-electrode distance using high-graduation micromanipulators;
(e) a highly focused electric field of a strength sufficient to obtain electroporation is applied between the electrodes.
The cell structure can be any kind of cell or cell-like structure, such as a cell in a primary cell culture, a cell in a tissue slice or a tissue, an in vivo cell, a liposome, or an intracellular cell structure, such as an organelle,.
The method according to the invention may be used either for transferring solutes from an extracellular medium into a permeabilised cell structure, or for transferring solutes entrapped in the cell structure out to the extracellular medium. The method according to the invention may also be used for transferring a substance into or out from an organelle, even when the organelle is located inside a cell.
The method is well-suited for the study of cellular migration, proliferation, growth, and differentiation, as well as a multitude of biochemical and biophysical events. It also opens up new possibilities for highly spatially resolved distribution of nanoparticles, drugs, genes and different biochemical markers, such as dyes into single cells or organelles both isolated and in situ. The method may be useful in clinical applications as a vehicle to administer drugs and genes to patients.
The method may also be useful for biosensor applications. In particular, a single cell can be placed in a permeabilising ac or dc field, thereby allowing cell-impermeant molecules that affect intracellular chemistry, including activation of receptors present on the surface of various organelles to be activated. In this way a compound library that acts on intracellular chemistry can be screened for biological activity. The compounds of interest can then be added to the cell solution using a perfusion system or a syringe. In a special case the compounds of interest can be delivered by a fused silica electrophoresis capillary of narrow inner dimensions. Because the electrophoresis capillary is connected to a voltage source, the electrophoresis capillary can be viewed as a liquid-filled electrode. If the outlet end of the electrophoresis capillary is placed close enough to the cell membrane, and an electric field strength sufficient to cause dielectric membrane breakdown is applied, compounds injected into the capillary will cross the cell-membrane barrier and enter the cell interior where they can act on intracellular receptors and intracellular chemistry. Because electrophoresis is a chemical separation technique, it can be used as a fractionation and screening method for biologically important compounds.
The characterising features of the invention will be evident from the following description and the appended claims.